Informace o publikaci
Multimeric structure of a subfamily III haloalkane dehalogenase-like enzyme solved by combination of cryo-EM and x-ray crystallography
| Autoři | |
|---|---|
| Rok publikování | 2023 |
| Druh | Článek v odborném periodiku |
| Časopis / Zdroj | Protein Science |
| Fakulta / Pracoviště MU | |
| Citace | |
| www | https://onlinelibrary.wiley.com/doi/10.1002/pro.4751 |
| Doi | http://dx.doi.org/10.1002/pro.4751 |
| Klíčová slova | catalysis; cryo-EM; DhmeA; haloalkane dehalogenase; Haloferax mediterranei; multimerization; x-ray crystallography |
| Přiložené soubory | |
| Popis | Haloalkane dehalogenase (HLD) enzymes employ an SN2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well-characterized enzymes, but the mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD-like enzyme from the archaeon Haloferax mediterranei, by combining cryo-electron microscopy (cryo-EM) and x-ray crystallography. We show that full-length wild-type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring-like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three-dimensional reconstructions of an oligomeric species by single-particle cryo-EM. Moreover, we engineered a crystallizable mutant (DhmeA(Delta GG)) that provided diffraction-quality crystals. The 3.3 angstrom crystal structure reveals that DhmeA.GG forms a ring-like 20-mer structure with outer and inner diameter of similar to 200 and similar to 80 angstrom, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization, and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates. |
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